2.1. Sialyloligosaccharides in Cheese Waste Streams
Whey is a major by-product of cheese manufacturing, which, for environmental reasons, presents a difficult waste disposal problem. In the United States alone, fluid whey is being produced at a rate of about 62.6 billion pounds annually. Whey is typically composed of about 5 wt. % lactose, 1 wt. % protein and about 0.5 wt. % salts, where the balance of the mixture is water. A major effort by many cheese making countries is presently underway to develop uses for this commodity, which formerly was considered a cheese processing waste product.
Although the protein concentrate obtained by ultrafiltration of whey has become a valuable commodity in the food industry and has found applications in animal feed, fertilizer, fermentation, and food filler, the majority of the resulting lactose-rich ultrafiltered permeate is still considered a disposable fraction.
Recently, several sialyloligosaccharides have been found to have valuable application as pharmaceutics. See, e.g. U.S. Pat. No. 5,270,462 to Shimatani et al. Sialyllactose has been shown to neutralize enterotoxins of various pathogenic microbes including Escherichia coli, Vibrio cholerae and Salmonella. See, e.g. U.S. Pat. No. 5,330,975 to Hiroko et al. It has also been shown that .alpha.(2-3) sialyllactose (.alpha.-Neu5Ac-(2-3)-Gal-.beta.-(1-4)-Glc) interferes with colonization of Helicobacter pylori and thereby prevents or inhibits gastric and duodenal ulcers. See e.g. U.S. Pat. No. 5,514,660 to Zopf et al. Sialyllactose has additionally been proposed to inhibit immune complex formation by disrupting occupancy of the Fc carbohydrate binding site on IgG and to be useful in treating arthritis. See, e.g. U.S. Pat. No. 5,164,374 to Rademacher et al.
To date, commercially available sialyloligosaccharides have been very expensive due to their low quantity in natural sources. For example, .alpha.(2-3) sialyllactose and .alpha.(2-6) sialyllactose isolated from bovine colostrum, is sold for $75.60 and $83.30 per milligram, respectively (Sigma Chemical Company, 1997).
A focused effort has been directed toward harvesting sialyloligosaccharides from the vast supply of whey made available as a cheese processing waste product. Processes for isolating sialyloligosaccharides have utilized such techniques as ultrafiltration, ion-exchange resins and phase partition chemistry. U.S. Pat. No. 4,001,198 to Thomas and U.S. Pat. No. 4,202,909 to Pederson; U.S. Pat. No. 4,547,386 to Chambers et al.; U.S. Pat. No. 4,617,861 to Armstrong; U.S. Pat. Nos. 4,971,701 and 4,855,056 to Harju et al.; U.S. Pat. No. 4,968,521 to McInychyn; U.S. Pat. No. 4,543,261 to Harmon et al.; U.S. Pat. Nos. 5,118,516 and 5,270,462 to Shimatani; J P Kokai 01-168,693; J P Kokai 03-143,351; J P Kokai 59-184,197; J P Kokoku 40-1234; J P Kokai 63-284,199 and Japanese Patent Publication No. 21234/1965, each of which is herein incorporated by reference in its entirety. Yields of up to 6 grams of .alpha.(2-3) sialyllactose sialyloligosaccharide per kilogram of cheese processing waste stream have been reported. U.S. Pat. No. 5,575,916 to Brian et al. which is herein incorporated by reference in its entirety.
2.2. Sialidases and Sialyltransferases
Sialic acids are 9-carbon carboxylated sugars which generally occur as the terminal monosaccharides in oligosaccharide chains. In mammalian cells, sialic acids are most frequently linked to .beta.-galactose with an .alpha.(2-3) linkage, and to N-acetylglucosamine and N-acetylgalactosamine with an .alpha.(2-6) linkage. Cross et al., 1993, Annu. Rev. Microbiol. 47:385-411.
Sialidases catalyze the removal of sialic acid residues from the oligosaccharide chain. Due to the wide variety of substitutions which may occur at various carbons of the sialic acid molecules, there are at least 39 different species of sialic acids. Colli, W., 1993, FASEB J. 7:1257-1264. Generally, sialidases exhibit substrate specificity for specific forms of sialic acid linkages. Viral sialidases cleave .alpha.(2-3) glycosidic bonds more efficiently than .alpha.(2-6) bonds, but bacterial sialidases are not as specific. Cross et al., 1993, Annu. Rev. Microbiol. 47:385-411 (citing Corfield et al. 1982, Sialic Acids: Chemistry, Metabolism and Function, Vol. 10, New York: Springer-Verlag, pp. 195-261). At low enzyme concentrations, bacterial sialidases exhibit a preference for cleaving .alpha.(2-3) or .alpha.(2-6) glycosidic bonds. Cross et al., 1993, Annu. Rev. Microbiol. 47:385-411.
CMP-sialyltransferases catalyze the transfer of cytidine monophosphate-sialic acid (CMP-sialic acid) residues to acceptor molecules. Although many sialidases exhibit at least some substrate specificity, CMP-sialyltransferases act on specific substrates. Mammalian CMP-sialyltransferases are generally found in the Golgi, however, there is evidence that there may be cell-surface associated CMP-sialyltransferases as well. Cross et al., 1993, Annu. Rev. Microbiol. 47:385-411 (citing Roth et al., 1971, J. Cell Biol. 51:536-547; Shur, 1991, Glycobiology 1:563-575; Yogeeswaran et al., 1974, Biochem. Biophys. Res. Commun. 59:591-599).
2.3. Trypanosoma Cruzi .alpha.(2-3)-Trans-Sialidase
Trypanosoma cruzi (Order Kinetoplastida) is the intracellular parasite responsible for Chagas diseage, throughout Iberoamerican countries. Chagas disease primarily affects nerve and muscle cells. One serious manifestation of Chagas disease is a chronic progressive fibrotic myocarditis. Colli, 1993, FASEB J. 7:1257-1264. Approximately 16-18 million people are infected with T. cruzi. Colli, 1993, FASEB J. 7:1257-1264.
T. cruzi invades a broad range of host cells, and a considerable amount of research has focused on the surface molecules in order to determine which molecules may be involved in parasite/host interaction. Colli, 1993, FASEB J. 7:1257-1264. One surface molecule which has generated a great deal of interest is the .alpha.(2-3)-trans-sialidase. This molecule has the capability of catalyzing both the removal of sialic acid from a donor saccharide-containing molecule (sialidase activity) and catalyzing the transfer of the sialic acid to an acceptor saccharide-containing molecule (trans-sialidase activity). Schankman et al., 1992, J. Exp. Med. 175:567-575. The gene encoding T. cruzi trans-sialidase has been cloned and characterized at the molecular level.
The T. cruzi .alpha.(2-3) trans-sialidase catalyzes the transfer of sialic acid from a donor terminal .beta.-galactosyl sialoglycoconjugate to a terminal .beta.-galactose on an acceptor molequle, Collit W., 1993, FASEB J. 7:1257-1264. T. cruzi .alpha.(2-3) trans-sialidase does not use CMP-sialic acid as a substrate and prefers sialyl .alpha.(2-3)-linked to .beta.-galactosyl residues as sialic acid donor molecules over sialyl .alpha.(2-6)-, .alpha.(2-8)-, and .alpha.(2-9)-linked sialic acids. Schenkman et al., 1994, Annu. Rev. Microbiol. 48:499-523. Furthermore, T. cruzi .alpha.(2-3) trans-sialidase cannot use free sialic acid as a substrate. Vandekerckhove et al. 1992, Glycobiology 2:541-548. The T. cruzi .alpha.(2-3) trans-sialidase has a broad pH optimum centered at 7.0. Cross et al., 1993, Annu Rev. Microbiol. 47:385-411.
More detailed analysis of the .alpha.(2-3) trans-sialidase has revealed that the amino-terminal portion of the protein is responsible for the .alpha.(2-3) trans-sialidase activity. Campetella et al., 1994, Mol. Biochem. Parasitol. 64:337-340; Schenkman et al., 1994, J. Biol. Chem. 269:7970-7975. It has also been determined that there are at least two critical amino acid residues: Tyr.sup.342 and Pro.sup.231 of the .alpha.(2-3) trans-sialidase appear to be required for full .alpha.(2-3) trans-sialidase activity. Cremona et al., 1995, Gene 160:123-25. The importance of Tyr.sup.342 is demonstrated by the fact that naturally occurring variants of the T. cruzi .alpha.(2-3) trans-sialidase which have a Tyr.sup.342.fwdarw.His substitution, lack .alpha.(2-3) trans-sialidase activity. Uemura et al., 1992, EMBO J. 11:3837-3844.
Trans-sialidase activity has also been discovered in Trypanosoma brucei, the causative agent of African Sleeping Sickness, Endotrypanum spp. and in Pneumocystis carinii. Like the T. cruzi .alpha.(2-3) trans-sialidase, the T. brucei trans-sialidase has a pH optimum of 7.0. However, unlike the T. cruzi trans-sialidase, which is expressed during the trypomastigote stage, the T. brucei trans-sialidase appears to be expressed only during the procyclic stage of the parasite life cycle, when the parasite resides in the midgut of its insect vector (Glossina spp., the "tsetse fly"). Cross et al., 1993, Annu Rev. Microbiol. 47:385-411.
2.4. Sialyllactose Production
A variety of methods for enzymatically producing sialylated oligosaccharides have been described.
U.S. Pat. No. 5,374,541 to Wong et al., describes a method for producing sialyloligosaccharides. According to this method, .beta.-galactosidase is used to form .beta.-galactosyl glycosides in the presence of CMP-sialic acid and .alpha.(2-3)- or .alpha.(2-6)-CMP-sialyltransferases to form sialylated oligosaccharides. This method does not use .alpha.(2-3) trans-sialidase.
U.S. Pat. No. 5,409,817 to Ito et al., discloses a three enzyme process for producing .alpha.(2-3) sialylgalactosides. According to this process, CMP-sialyltransferases transfer sialic acid from CMP-sialic acid to acceptor molecules, these acceptor molecules become donor molecules for Trypanosoma cruzi .alpha.(2-3) trans-sialidase, and CMP-sialic acid is regenerated in the system through the action of CMP-sialic acid synthetase and added free sialic acid.
The process described in U.S. Pat. No. 5,409,817 to Ito et al. specifically requires the addition of free sialic acid. The free sialic acid is converted to CMP-sialic acid by CMP-sialic acid synthetase, and the sialic acid moiety is transferred to an acceptor molecule by CMP-sialyltransferase. According to the disclosure of Ito et al., the formation of these sialylated acceptor molecules is required to drive the .alpha.(2-3) trans-sialidase reaction forward.
In addition to free sialic acid, the method of Ito et al., also requires the presence of three enzymes including CMP-sialic acid synthetase and CMP-sialyltransferase. Further, dairy sources and cheese processing waste streams do not contain CMP-sialic acid synthetase.
2.5. Expression of Transgenes in Milk
Numerous foreign proteins have successfully been transgenically expressed in the milk of livestock. Most of this work has focused on the expression of proteins which are foreign to the mammary gland. Colman, A., 1996, Am. J. Clin. Nutr. 63:639S-645S. To date, milk specific expression of transgenic livestock has been achieved through operably linking regulatory sequences of milk-specific protein genes to the target protein-encoding gene sequence, microinjecting these genetic constructs into the pronuclei of fertilized embryos, and implanting the embryos into recipient females. See e.g. Wright et al., 1991, Biotechnology (NY) 9:830-834; Carver et al., 1993, Biotechnology (NY) 11:1263-1270; Paterson et al., 1994, Appl. Microbiol. Biotechnol. 40:691-698. Proteins that have been successfully expressed in the milk of transgenic animals, include: .alpha.1-antitrypsin (Wright et al., 1991, Biotechnology (NY) 9:830-834; Carver et al., 1993, Biotechnology (NY) 11:1263-1270); Factor IX (Clark et al., 1989, Biotechnology (NY) 7:487-492); protein C (Velander et al., 1992, Proc. Natl. Acad. Sci. USA, 89:12003-12007); tissue plasminogen activator (Ebert et al., 1991, Biotechology (NY) 9:835-838); and fibrinogen. While most of these transgenes express proteins that supplement the composition of milk, very few, if any of the expressed proteins interact directly with the components of milk to alter the natural milk composition. There is a need for methods providing for the large scale production of .alpha.(2-3) sialyloligosaccharides, such as .alpha.(2-3) sialyllactose, which have commercial and/or therapeutic valve.